Lactobacillus paracasei for the production of conjugated linoleic acid, nutritional and pharmaceutical preparations containing it and uses thereof

ABSTRACT

The present invention refers to a new strain belonging to the species  Lactobacillus paracasei  able to convert linoleic acid into conjugated linoleic acid. The present invention refers also to nutritional or food preparations and/or pharmaceutical compositions, comprising the strain  Lactobacillus paracasei , useful in the treatment and/or prevention of pathologies and/or physiological states related to conjugated linoleic acid deficiency or in the cases wherein the use of a probiotic is beneficial.

STATE OF THE ART

The present invention relates to a new strain belonging to the speciesLactobacillus paracasei, called LMG S-26420, able to convert linoleicacid (LA) into conjugated linoleic acid (CLA), useful in the treatmentand prevention of diseases and/or physiological states related toconjugated linoleic acid deficiency or in cases where it is recommendedthe use of a probiotic, to processes for obtaining it and topharmaceutical or nutritional compositions containing it.

The strain Lactobacillus paracasei LMG S-26420 is also useful in thetreatment and prevention of all disorders where the assumption of aprobiotic is beneficial, for example for restoring and maintaining theintestinal flora balance.

The term CLA refers to a mixture of positional and geometrical isomersof linoleic acid (LA), wherein the double bonds are conjugated invarious positions, thus generating cis- and trans-isomers.

At least 16 CLA isomers are known and the isomers of major biologicalinterest are the isomers c9-t11 and t9-t11. The isomer c9-t11 is thepredominant isomer in diets, and it has proved to be important becauseit is involved in many biological processes and is incorporated in thephospholipidic fraction of animal tissues, which are fed with mixturesof CLA isomers.

The CLA production in human organism is substantially irrelevant and itssupply is therefore entrusted to the ingestion of dairy products andmeat through diet or supplements containing probiotic bacteria.

Foods produced by ruminants are the major CLA source for humans. Theyare intermediates in the bio-hydrogenation of linoleic acid and it isgenerally accepted that CLA in ruminants is originated by the incompletebio-hydrogenation of the unsaturated acids of the linoleic acid byruminal bacteria.

CLA is introduced in the organism through a diet with milk, fish, meatand dairy products, and its beneficial effects are correlated to dailyassumption levels of about 3 g/day. An unbalanced diet gives an averageCLA assumption of 0.35 g/day and, considered its beneficial effects forthe organism, this deficiency must be compensated through the assumptionof probiotics converting LA into CLA to provide useful dosages.

The biological activities of the conjugated linoleic acids are largelyattributed to the action of isomers c9-t11 and t10-c12; the majorbiological activities linked to anti-carcinogenic effects are attributedto the isomer c9-t11, while the isomer t10-c12 is involved in the lipidmetabolism of the human body.

CLA has important biological properties and is useful for human andanimal health, such as e.g. in intestinal inflammatory pathologies,diarrhea and colon inflammation, in increasing body mass, in increasingthermogenesis, in oxidative stress protection, in tumour prevention, inautoimmune diseases, inflammatory diseases, diabetes andatherosclerosis.

Ecker J. et al. in Biochem. Bioph. Res. Comm. 388, 2009, 660-666 reportthat the isomer c9-t11 activates the target gene LXR involved in thedevelopment and progress of atherosclerosis. Ogawa J. et al in Appl.Env. Microbiol 2001, 67, 1246 describe the production of specific CLAisomers from linoleic acid by lactobacilli, Lactobacillus acidophilus inmicro-aerophilic conditions, assuming that the hydroxy acid10-hydroxy-cis-12-octadecanoid is the intermediate for this conversionand that the conversion involves more than one step.

Ogawa J. et al. in J. Bioscience Bioeng. 100 (4), 355 (2005) report theconversion of LA into CLA by bifidobacteria and lactobacilli with theobtainment of a mixture of isomers. When bifidobacteria are used, theproduction of CLA isomers varies from about 3 to about 400 mg per litreof culture, while when lactobacilli are used, the productivity variesfrom about 100 mg to about 4 g per litre of culture. When Lactobacilluscasei are used, the productivity never exceeds 1 g per litre of culture.

Rosberg-Cody E. et al in Appl. Env, Microbiol 70 (8) 4635, 2004 describethe isolation of bifidobacteria strains from new-born faecal material,useful for the production of CLA, and suggest the use of those bacteriain supplements for new-borns at risk of necrotizing enterocolitis.

Also Coakley M. et al. in J. Appl. Microbiol. 94, 138 (2003) describethe ability of lactobacillus, lactococcus and bifidobacteria to convertLA into CLA, and demonstrate that bifidobacteria strains have a higherability of conversion of LA into CLA, with conversion percentages up toalmost 65%.

Alonso L. et al. in J Diary Sci. 86, 1941 (2003) describe the conversionof LA into CLA by Lactobacilli casei and Lactobacilli acidophilus ofhuman intestinal origin in media added with different concentrations oflinoleic acid. At concentrations of 0.02% of linoleic acid, the maximumCLA concentration obtained with Lactobacillus casei is about 110 mg perlitre of culture and the amount of the isomer of biological interestc9-t11 ranges from 60 to 85 mg per litre of culture.

Gustavo A. et al, in Biosci. Biotechnol. Biochem., 77 (3), 648-650,2013, describe a rapid and simpler method to screen conjugated linoleicacid (CLA)-producing bacteria isolated from cow milk. One strainresembling a L. Paracasei converted free linoleic acid to total CLA in apercentage greater than 85%. However the percentage of conversion in CLAisomer of biological interest c9,t11 is only about 18%.

Oguz Gursoy et al, in International Journal of Food Sciences andNutrition, 63(5), 610-615, describe the effect of using differentprobiotics cultures on conjugated linoleic acid concentration and fattyacid composition of cheese. They report that the probiotic differencesand storage process have not affected the CLA contents of the samplesstatistically. The increment of CLA content of cheese samples is due tolipolysis of the free linoleic acid by lactic acid bacteria.

The assumption of probiotic products comprising lactobacilli is alsouseful for maintaining the intestinal bacterial flora balance and is auseful tool in the prevention and treatment of dysbiosis in general,where the balance between bifidobacteria and lactobacilli is essential.

Given the low amount of CLA introduced with the diet, and the dosageuseful in all pathologies and disorders connected to a CLA deficiency,there was the need to have available bacteria converting LA into CLA forobtaining daily dosages useful for the health of individuals and for thetreatment of all pathologies where CLA isomers are beneficial.

Bifidobacteria convert LA into CLA, but bifidobacteria cultures have thedisadvantage of having low productivity yields, meant as number ofbacteria per litre of culture, and therefore it is not easy to obtainthem in large amounts in industrial scale processes.

Therefore, there was the need to have a bacterial strain able to convertLA into CLA obtainable by bacterial cultures with high productivity tobe used in pharmaceutical or nutritional preparations useful in thetreatment or prevention of all disorders and pathologies related to aCLA deficiency.

There was also the need to have a bacterial strain belonging to thegenera Lactobacillus, able to convert LA into CLA isomers beneficial forhumans or animals. Moreover, there was also the need to have alactobacillus for all disorders or pathologies associated with amicrobial imbalance on the body surface. A strain belonging to thelactic genus can be ingested in association with bacteria of the genusBifidus to promote the microbial balance of human or animal flora.

The bacteria belonging to the genus Lactobacillus are among those withthe highest productivity; therefore, they are preferable to thosebelonging to the genus Bifidobacterium to convert LA into CLA.Lactobacilli can be obtained by industrial processes with higher yieldsthan bifidobacteria, and can be used for the production of nutritionalor pharmaceutical compositions for the administration in human oranimal.

Among these, lactobacilli able to convert LA into isomers c9-t11 andt9-t11 of CLA to a greater extent than the other isomers are preferred.c9-t11 is a major isomer because it is involved in cell membranephospholipids and is the predominant isomer in diets. These isomers havebeneficial effects on humans and animals, and in particular the isomert9-t11 has anti-proliferative and anticancer properties. Ecker J et al.in Biochem. Biophysical. Res. Comm. 388, 660, 2009 report that theisomer t9-t11 of CLA is a powerful agonist of macrophagic LXR, linked tothe inflammation induction processes, and it has an important role inthe reduction of arteriosclerotic processes in animal models.

The present invention describes a strain belonging to the genusLactobacillus, named Lactobacillus paracasei LMG S-26420, able toconvert LA into CLA with a conversion percentage higher than the otherlactobacilli strains known in the art. Moreover, the strain LMG S-26420of the present invention is characterized by converting LA into CLA,wherein the mixture of isomers c9-t11 and t10-c12 is prevalent ifcompared to the other isomers.

The strain is obtained with bacterial cultures characterized by aproductivity higher than 4 g per litre of culture and it can be obtainedin lyophilic form.

The strain is stable and the product of the bacterial culture can bestored for long periods at temperatures lower than 0° C. or for periodslonger than 6 months at 4° C. in lyophilic form. The strain LMG S-26420can be comprised in nutritional or food preparations or inpharmaceutical compositions useful for the treatment and/or theprevention of disorders or pathologies linked to a CLA deficiency and inall disorders wherein a probiotic assumption is beneficial for humans oranimals.

SUMMARY

The present invention describes a new strain belonging to the genusLactobacillus, named Lactobacillus paracasei, filed at the BelgianCoordinated Collections of Microorganisms BCCM/LMG BacteriaCollection-Microbiology Laboratory-Ghent University on Apr. 15, 2011with the number LMG S-26420.

The Lactobacillus Paracasei LMG S-26420, is characterized in that itconverts linoleic acid in conjugated linoleic acid in a percentagehigher than 30%.

The strain Lactobacillus paracasei LMG S-26420 is characterized byconverting linoleic acid (LA) into conjugated linoleic acid (CLA), in apercentage higher than 30% if compared to the starting LA, wherein thebiological isomers with biological activity c9-t11 and t9-t11 are in apercentage higher than 30% if compared to the other CLA isomers.

The strain Lactobacillus paracasei LMG S-26420 is characterized by theproduction of high concentration of CLA.

The strain Lactobacillus paracasei LMG S-26420 is obtained by bacterialcultures characterized by a productivity higher than 4 g per litre ofculture and a yield of Colony Forming Units (CFU) higher than 1×10⁹ permillilitre of culture.

The strain Lactobacillus paracasei LMG S-26420 is characterized by beingstable: it can be stored at temperatures lower than 0° C. and forperiods longer than 6 months at 4° C. in lyophilic form.

The strain lyophilic Lactobacillus paracasei LMG S-26420 ischaracterized by an amount of living cells higher than 1×10¹⁰, inparticular from about 1×10¹⁰ to about 7×10¹⁰ unit per gram of lyophilicproduct.

It is an object of the present invention the process for the productionof the strain Lactobacillus paracasei LMG S-26420 in bacterial culture,wherein the inoculum of the strain LMG S-26420 has a concentration from0.1 to 10% (v/v) in a culture medium at a temperature comprised between30° C. and 37° C., at pH values comprised between 4.5 and 7.5, for aperiod comprised between 6 and 15 hours. The biomass is separated and itcan be preserved at temperatures lower than 4° C. or submitted tolyophilisation processes.

The described process leads to the obtainment of the strainLactobacillus paracasei LMG S-26420 with a number of colony-formingunits comprised between 1×10⁹ and 5×10⁹ CFU per millilitre of cultureand with a biomass in an amount comprised between 10 and 20 grams perlitre of culture.

The process comprising the lyophilisation leads to the obtainment of thestrain LMG S-26420 in solid form. In the presence of cryoprotectants,the strain is obtained with yields of colony-forming units (CFU) higherthan 50% and the lyophilic strain LMG S-26420 includes an amount ofcells higher than 1×10¹⁰ per gram of lyophilic product.

Objects of the invention are nutritional or food preparations andpharmaceutical compositions comprising an amount of Lactobacillusparacasei LMG S-26420 in lyophilic form corresponding to an amount ofliving cells from about 1×10⁸ to about 5×10₁₁.

Nutritional or food preparations and pharmaceutical compositionscomprising Lactobacillus paracasei LMG S-26420 can be in form ofsachets, tablets or capsules. The nutritional or food preparations andthe pharmaceutical compositions can comprise prebiotics, vitamins,mineral salts and pharmaceutical or nutritional excipients.

The prebiotics are comprised in Lactobacillus paracasei LMG S-26420compositions, selected from the group consisting offructo-oligosaccharides, inulines, galacto-oligosaccharides,xilo-oligosaccharides, isomalto-oligosaccharides and vitamins selectedfrom the group comprising the vitamins of the E and B complex.

The nutritional or food preparations and pharmaceutical compositionscomprising Lactobacillus paracasei LMG S-26420 can comprisebifidobacteria.

The compositions comprising Lactobacillus paracasei LMG S-26420 areuseful in the treatment and prevention of pathologies and/orphysiological states related to the deficiency of conjugated linoleicacid and in all other cases wherein the use of a probiotic is useful andhas a beneficial effect.

In particular, the compositions comprising Lactobacillus paracasei LMGS-26420 are useful in the treatment and prevention of pathologies and/orphysiological states related to a CLA deficiency, such as e.g. inintestinal inflammatory pathologies, as diarrhoeal and coloninflammation; in increasing body mass, in increasing thermogenesis, inoxidative stress protection, in tumour prevention, in autoimmunediseases, diabetes and atherosclerosis.

The compositions comprising Lactobacillus paracasei LMG S-26420 withbifidobacteria, object of the present invention, are useful in thetreatment of all disorders related to bacterial dysbiosis.

The compositions comprising Lactobacillus paracasei LMG S-26420 areuseful in all physiological states where the intestinal bacterial florabalance of humans and animals must be kept unaltered.

DESCRIPTION OF THE INVENTION

A pure bacterial culture of the strain belonging to the Lactobacillusparacasei was filed at the Belgian Coordinated Collections ofMicroorganisms-BCCM/LMG Bacteria Collection-MicrobiologyLaboratory-Ghent University with the number LMG S-26420.

The present invention refers to a new strain of Lactobacillus paracaseiLMG S-26420, to pharmaceutical, nutritional or food compositionscomprising said strain and to their use in the treatment and preventionof pathologies and/or physiological states related to the deficiency ofconjugated linoleic acid or when the use of a probiotic is suggested.

The strain Lactobacillus paracasei LMG S-26420 has been isolated fromthe vaginal bacterial flora of a healthy woman, selected among manyother strains of lactobacilli simultaneously isolated from the samesource and from other types of biological samples and among otherstrains of the same species, because it has shown an ability to convertlinoleic acid (LA) into conjugated linoleic acid (CLA). Thischaracteristic makes this strain a probiotic agent useful for human andanimal use.

Lactobacillus paracasei LMG S-26420 is characterized by converting LAinto CLA in a percentage higher than 30% determined by chromatographicmethod.

Lactobacillus paracasei LMG S-26420 converts LA into isomers of CLA withbiological activity, in particular in the isomers c9-t11 and t9-t11. Ina particular aspect of the invention, Lactobacillus paracasei LMGS-26420 converts LA into the isomers c9-t11 and t9-t11 of CLA in apercentage higher than 30%.

To evaluate the ability of the strain Lactobacillus paracasei LMGS-26420 to convert LA into CLA, three different methods were used: theOgawa method, the Liu method and the chromatographic method.

The use of the Ogawa J. method described in Appl. Environ. Microbiol67(3): 1246-1252, 2001 allows determining the ability of the strainLactobacillus paracasei LMG S-26420 to convert LA into CLA in a suitableculture medium by adding LA to the bacterial culture in order tometabolically adapt the cells.

In a first case, variable concentrations of LA, in a range from 0.01mg/ml to 1 mg/ml, are added to a series of cell cultures of the strainLMG S-26420 and cells are incubated for a period from 1 to 4 days attemperatures from 30° to 40° C. The final product is centrifuged and aconstant concentration of LA, equal to 5 mg/ml, was added to the cells.The cell cultures are incubated at a temperature ranging from 30° to 40°C., for a period from 40 to 80 hours and then centrifuged to remove thesupernatant. To evaluate the ability of a strain to convert LA into CLA,the cells are resuspended in water and CLA is determined byspectrophotometric method.

In a second case, a constant concentration of LA equal to 5 mg/ml isadded to a series of cell cultures of Lactobacillus paracasei LMGS-26420 and the biomass obtained at the end of the culture is incubatedwith variable concentrations of LA in a range from 0.05 mg/ml to 0.4mg/ml.

In both cases, Lactobacillus paracasei LMG S-26420 shown to be effectivein converting LA into CLA, in particular in a percentage higher than30%.

The use of the Liu P. method as described in Biomed. & Biotechnol.12811, 923-930, 2011, comprises the inoculation of the strain LMGS-26420 in an adequate culture medium with LA concentrations from 0.05to 1 mg/ml. The cultures are incubated for a period from 1 to 3 days attemperatures from 30° C. to 40° C. and then centrifuged. The CLAconcentrations is determined by means of spectrophotometric method.

The trials carried out according to the Ogawa and Liu methods showed theability of the strain Lactobacillus paracasei LMG S-26420 to convert LAinto CLA. However, it is not possible to evaluate the percentage ofconversion into CLA isomers with these methods, in particular if thestrain is able to convert LA into CLA isomers having the strongestbiological activity.

The chromatographic method, in turn, allows the separation andquantification of CLA geometrical isomers, isomers trans-trans,trans-cis and cis-cis, in particular isomers cis9-trans 11, trans 10-cis12 and trans 9-trans 11.

The obtained CLA isomers were determined by using silver-ionhigh-pressure liquid chromatography (HPLC) with a diode array detectorand a UV detector at 234 nm.

The chromatographic method demonstrates that the strain LMG S-26420converts LA into CLA with a percentage higher than 30%.

The chromatographic method demonstrates that the strain LMG S-26420converts LA into CLA with a percentage from 30% to 50%, wherein thepercentage of isomers with biological activity, c9-t11 and t9-t11, areprevalent if compared to the other isomers. In particular, the strainLMG S-26420 converts LA into CLA isomers c9-t11 and t9-t11, whereintheir percentage is 40% higher than all other CLA geometrical isomers.

The strain LMG S-26420 is obtained by cell cultures characterized bygiving a number of colony forming units (CFU) higher than 1×10⁹ permillilitre and an amount of solid mass higher than 4 g per litre ofculture. The obtained new bacterial strain is stable at temperatureslower than 4° C. for longer periods and it can be lyophilised withprocesses that significantly preserve cell viability. The lyophilicproducts are stable for periods of time longer than 3 months attemperatures of 4° C. and 25° C.

The strain Lactobacillus paracasei LMG S-26420 produces an amount oflyophilised biomass higher than 4 g per litre of culture, in particularfrom 10 to 20 g per litre of culture. Considering the ability of thisstrain to convert LA into CLA in a percentage higher than 30%, it can bestated that the new strain Lactobacillus paracasei LMG S-26420 is ableto produce remarkable concentrations of CLA. For example, addingLactobacillus paracasei LMG S-26420 to solutions includingconcentrations of about 500 mg/litre of linoleic acid leads to obtainconcentrations higher than 250 mg/litre of conjugated linoleic acid.

Another advantage of the present invention is to have a new strainbelonging to the genus Lactobacillus for the conversion of LA into CLAisomers with high conversion yields useful to be included in nutritionalor food preparations or in pharmaceutical compositions.

The new strain Lactobacillus paracasei LMG S-26420 can be comprised incompositions also comprising other bacterial strains, belonging to thesame genus or not, like bifidobacteria.

The compositions comprising LMG S-26420 and other bacterial strainsadvantageously generate a heterogeneous probiotic population, useful tomaintain the intestinal bacterial flora balance.

The bacterial cultures object of the present invention were producedfirst in laboratory scale and then in industrial scale. The bacterialculture of LMG S-26420 was obtained by means of a fermentation processin a period from 6 to 12 hours at temperatures ranging from 30° C. to40° C. in a medium named MRS® (De Man, Rogosa & Sharpe) containing asmain ingredients yeast extract, mixtures of peptones and glucose inaddition to potassium salts, ammonium, magnesium and manganese.

Starting from the primary cultures, many expansion phases were conductedto increase the number of cells per volume of culture of the pure strainin order to obtain the so called “mother cultures”, that were used asinoculum for the industrial production of the bacterial cultures of theprobiotic LMG S-26420.

The industrial process for the production of Lactobacillus paracasei LMGS-26420 comprises the following steps:

-   -   inoculating the strain LMG S-26420 with volumes of mother        cultures, in a volumetric percentage from 0.1 to 10% and        fermenting in an adequate medium of culture at 37° C. and pH        from 4.5 to 7.5, for a period from 8 to 15 hours;    -   separating the bacterial biomass from the culture broth by        centrifugation.

The biomass can be frozen or submitted to lyophilisation processes afteradding suitable cryoprotectants, selected among soluble carbohydrates,which are useful to maintain the cell viability during thelyophilisation processes.

The strain LMG S-26420 is obtained by fermentation processescharacterized by producing a number of living cells higher than 10⁹living cells per ml of culture and a lactobacilli biomass higher than 4g per litre of culture.

The strain LMG S-26420 is obtained with a productivity from 1×10⁹ to7×10⁹ living cells per ml of culture and has a yield of dried productfrom 10 to 20 g per litre.

The cell viability is determined using bacterial counting methods knownto the person skilled in the art.

The strain LMG S-26420 obtained from the described cultures can belyophilised to be easily stored and to be included in alimentary, foodor pharmaceutical preparations.

The lyophilisation process was executed in the presence ofcryoprotectants chosen among soluble carbohydrates, such as for exampletrealose and cyclodextrins or a mixture of them. The describedlyophilisation process is characterized by obtaining a lyophilic strainLMG S-26420 with a yield in vital cells higher than 50% than before thelyophilisation process. The strain LMG S-26420 is characterized bycomprising an amount of living cells per gram of lyophilic producthigher than almost 1×10¹⁰, in particular from almost 1×10¹⁰ to almost7×10¹⁰ per gram of lyophilic product. This confirms that the strainLactobacillus paracasei LMG S-26420 is characterized by a highproductivity, can be lyophilised and maintained as lyophilic productwith the certainty of preserving cell viability.

The strain Lactobacillus paracasei LMG S-26420 obtained with thedescribed cell cultures and lyophilised in presence of cryoprotectantsselected among soluble carbohydrates or mixtures thereof, ischaracterized by a water activity (a_(w)) lower than 0.6, a value belowwhich the proliferation of the majority of bacteria and mildews isinhibited. The water activity is a value obtained by measuring thepartial steam pressure in a substance divided by the partial pressure ofwater, and is obtainable with detectors directly giving those values.

As described by Ryser, E. T. et al in Listeria, Listeriosis and FoodSafety (3rd ed.). CRC Press. 173-174, (2007), water activity valueslower than 0.6 assure that the lyophilic preparation can be storedbecause it does not degrade because of microbial proliferation, which isone of the most dangerous causes of alimentary or food alterations.

The lyophilic products of the strain LMG S-26420s are characterized byhaving an elevated number of living cells per gram of lyophilic productand a low content of water activity, are useful to be stored for thepreparation of compositions or preparations in different formscontaining different probiotic amounts, with no limitation.

The LMG S-26420 bacterial strain in the solutions submitted to thelyophilisation processes is characterized by values of glass transitiontemperatures, T_(g), higher than 100° C., and in the presence of solublecarbohydrates, such as e.g. trealose, the values of glass transitiontemperatures are in a range from 100° C. to 120° C. These values confirmthat this strain can be submitted to lyophilisation and vacuum dryingprocesses up to temperatures of 100° C. without having the productconverted into a glassy state, with a consequent loss of its properties.

The glass transition temperature of frozen solutions T_(g)′ relating tothe preparations to be lyophilised is in a range from −15° to −30° C.and in the presence of soluble carbohydrates, as e.g. trealose, theglass transition temperatures are in a range from −20 to −30° C.

The lyophilisation process of the strain Lactobacillus paracasei LMGS-26420 described in Example 3 in the presence of cryoprotectants chosenamong soluble carbohydrates, in particular trealose, is characterized bya freezing step carried out at temperatures lower than −30° C. and asecondary drying carried out at temperatures lower than 100° C. Saidprocess leads to the obtainment of the strain LMG S-26420 in lyophilicform, which is characterized by maintaining its biological properties.

In particular, the lyophilisation process of the strain Lactobacillusparacasei LMG S-26420 described in the invention in the presence oftrealose maintains a cell viability higher than 30%, in particular 30%to 60%, if compared to the cell viability before lyophilisation.

The described lyophilic compositions of the strain Lactobacillusparacasei LMG S-26420 comprise soluble carbohydrates, such as trealoseand mannitol; this latter can be added to trealose-comprising lyophilicpreparations when a high lyophilic mass is requested, in particular tobe used in preparations with a low dosage of probiotic strain.

The solutions comprising Lactobacillus paracasei LMG S-26420 to belyophilised can comprise osmolytes chosen from the group consisting ofbetaine, sarcosine, glycerol, erythritol; salts selected from the groupconsisting of acetates, formiates or ammonium salts, useful to maintainpH values between 4 and 8 during the lyophilisation process.

The lyophilic strain Lactobacillus paracasei LMG S-26420 is stable at atemperature of 4° C. for a period longer than 3 months. In a particularaspect, Lactobacillus paracasei LMG S-26420 maintains more than 70% ofits cell viability after 6 months at the temperature of 4° C., and thewater activity remains with values lower than 0.6%.

The lyophilic product can therefore be prepared in large amounts and bestored for the preparation of compositions in solid or suspended form atdifferent probiotic dosages.

An aspect of the present invention refers to pharmaceuticalcompositions, nutritional or food preparations comprising variableamounts of the strain LMG S-26420 in lyophilic form in amounts rangingfrom 20 to 2500 mg. These compositions are characterized by comprisingan amount of living cells of LMG S-26420 from 1×10⁹ to 1×10¹¹ per gramof lyophilic product.

The preparations can be in a form useful for oral administration, suchas e.g. in sachets, tablets, capsules or liquid suspensions.

The compositions in form of tablets or capsules can comprise an amountof the strain Lactobacillus paracasei LMG S-26420 ranging from 20 to 800mg and the sachet compositions for liquid suspension can comprise anamount of Lactobacillus paracasei LMG S-26420 ranging from 20 mg to 10grams. The pharmaceutical or nutritional compositions can comprise anamount of Lactobacillus paracasei LMG S-26420 ranging from about 1×10⁹to 1×10¹¹ unity of cells.

The pharmaceutical, nutritional or food compositions comprisingLactobacillus paracasei LMG S-26420 can optionally comprise, but are notlimited to, prebiotics selected from the group consisting offructo-oligosaccharides, inulines, galacto-oligosaccharides,xilo-oligosaccharides, isomalto-oligosaccharides, resistant dextrin,polydextrose, arabinogalactans, resistant starch, dextrans, guar gum;amino acids; proteins; antioxidants; vitamins selected from the groupcomprising the vitamins of the E and B complex, together withpharmaceutically acceptable salts useful for the preparation of thedesired form.

The pharmaceutical, nutritional or food compositions comprisingLactobacillus paracasei LMG S-26420 can comprise also other bacteriaable to convert LA into CLA, in particular those belonging to the genusBifidobacterium. These compositions increase the conversion of LA intoCLA and favour the intestinal bacterial flora balance.

The compositions in form of sachets are prepared by mixing the strainLactobacillus paracasei LMG S-26420 in form of lyophilic products withselected excipients, such as e.g. oligosaccharides, selected from thegroup of fructo-oligosaccharides, inulines, galacto-oligosaccharides,xilo-oligosaccharides, isomalto-oligosaccharides and flavours,previously sieved. The homogeneous mixture is then divided in sachets.

The lyophilic form can be grinded or granulated before being added tothe pharmaceutical excipients chosen for the preparation of the desiredsolid forms.

The compositions in form of tablets can comprise diluents, ligands,disintegrants, lubricants, glidants and are prepared according to thetechniques known to the person skilled in the art.

Optionally, the compositions can comprise preservatives, antioxidants,buffering, colouring, flavouring and sweeting agents.

The compositions of the invention comprising the strain Lactobacillusparacasei LMG S-26420 are stable at temperature of 4° C. and 25° C. fora period of 1, 3 and 6 months with a complete recovery of the livingcells.

Another aspect of the invention refers to the use of pharmaceutical,nutritional or food compositions comprising the strain Lactobacillusparacasei LMG S-26420 for use in the treatment and prevention ofpathologies and/or physiological states wherein a probiotic is useful.These compositions are useful in the prevention and treatment ofpathologies and/or physiological states related to a CLA deficiency suchas, e.g. in intestinal inflammatory pathologies, diarrhea and coloninflammation, in increasing body mass, in increasing thermogenesis, inoxidative stress protection, in tumour prevention, in autoimmunediseases, inflammatory diseases, diabetes and atherosclerosis.

The compositions comprising the strain Lactobacillus paracasei LMGS-26420 are useful in the treatment and prevention of all disorders orpathologies connected to a microbial unbalance.

The compositions comprising the strain Lactobacillus paracasei LMGS-26420 are also useful as alimentary supplement in all diets poor ofmeat or milk products to obtain CLA concentrations beneficial forhumans.

EXAMPLES

The Examples refer to the strain Lactobacillus paracasei LMG S-26420exactly corresponding to the one filed at the Belgian CoordinatedCollection of Microorganisms, BCCM, LMG Bacteria Collection,Microbiology Laboratory, Ghent University, which confirmed the purityand viability of the strain and registered it with the number LMGS-26420. In particular, the examples refer to the characterization ofthe strain LMG S-26420, to its ability to convert LA into CLA, to itsproduction process in industrial scale and to compositions containingit.

Example 1

Determination of the Strain Ability to Convert Linoleic Acid (LA) intoConjugated Linoleic Acid (CLA)

To determine the ability of the strain Lactobacillus paracasei LMGS-26420 to convert LA into CLA, it has been evaluated a conditionwherein the inoculum was made in presence of LA, to stimulate bacterialcells pre-adaptation, and a condition without LA.

a) Determination According to the Ogawa Method

The quantitative determination of the ability of Lactobacillus paracaseiLMG S-26420 to convert LA into CLA was carried out according to theOgawa method described in Appl. Envr. Microbiol. 2001, 67, 1246.

The method is based on the inoculum of the strain in MRS® (Man, Rogosa,Sharpe) cultural medium comprising a mixture of peptones 18 g/l; yeastextract 4 g/l; glucose 20 g/l; tween-80 1 ml/l; potassium phosphate 2g/l; tri-ammonium citrate 2 g/l; anhydrous sodium acetate 3 g/l;heptahydrate magnesium sulphate 0.2 g/l; anhydrous magnesium sulphate0.034 g/l; agar 12 g/l.

The strain Lactobacillus paracasei LMG S-26420 at a concentration of 1%was inoculated in 15 ml of MRS® with LA at concentrations from 0.01 to0.4 mg/ml and the solutions were kept at a temperature of 37° C. for 3days at low stirring. The cultures were centrifuged and the supernatantwas eliminated. The pellet was washed with sterile water and the cellmass of about 20 mg was resuspended with 1 ml of solution of potassiumphosphate buffer 100 mM at pH 6.5 and 5 mg of LA in a complex withbovine albumin (BSA) were added to cell suspension, with a ratiocorresponding to 0.2 mg BSA/mg of LA. The cultures were then incubatedfor 48 and 72 hours and then centrifuged to eliminate the supernatant.The cells were resuspended in water and the CLA concentration wasspectrophotometrically determined by means of the Barret method,described in Appl. Environment Microbiol. 73(7), 2333, (2007). Thismethod comprises the extraction of the fatty acid fraction by theaddition of 2 ml of isopropanol to 1 ml of sample. The solutions werevigorously stirred, allowed to stand for 3 minutes and then 1.5 ml ofhexane were added. Organic phases were separated and dehydrated withanhydrous sodium sulphate and the CLA amount was determined byspectrophotometric reading at 233 nm. The concentrations were determinedby a calibration curve obtained with different concentrations of the CLAisomer c9-t11 at the same wavelength.

Table 1 reports the conversion percentages of LA into CLA by means ofthe strain Lactobacillus paracasei LMG S-26420, wherein thepre-incubation of the strain was carried out at different concentrationsof LA and the incubation of the washed cells was carried out with 5mg/ml of LA. Table 1 shows that, in the reported conditions, theconversion percentages of LA into CLA range from 1% to 7%.

TABLE 1 Cell Conversion LA Cell incubation CLA percentage concentrationincubation temperature concentration (%) of LA (mg/ml) time (h) (° C.)(mg/ml) into CLA 0.01 48 30 0.083 2 0.05 48 30 0.075 2 0.1 48 30 0.067 10.2 48 30 0.041 1 0.3 48 30 0.045 1 0.4 48 30 0.104 2 0.01 72 37 0.204 40.05 72 37 0.273 5 0.1 72 37 0.097 2 0.2 72 37 0.349 7 0.3 72 37 0.282 60.4 72 37 0.085 6

Under the conditions reported in Table 2, CLA is not present in thesupernatant.

Table 2 reports the results of the conversion of LA into CLA. Thepre-incubation was carried out with LA at a concentration of 0.05 mg/mland the washed cell mass was incubated at a variable concentration ofLA. CLA was determined on the supernatant and on the cell pellet.

TABLE 2 LA Cell CLA Conversion concen- Cell incubation concen-percentage tration incubation temperature tration (%) of LA (mg/ml) time(h) (° C.) Sample (mg/ml) into CLA 0.05 72 37 Cells — — 0.05 72 37Supernat. 0.013 25 0.1 72 37 Cells — 0.1 72 37 Supernat. 0.004 4 0.2 7237 Cells — 0.2 72 37 Supernat. — — 0.3 72 37 Cells — — 0.3 72 37Supernat. — — 0.4 72 37 Cells 0.007 2 0.4 72 37 Supernat. 0.005 1 0.5 7237 Cells 0.007 1 0.5 72 37 Supernat. 0.003 1 1 72 37 Cells 0.025 3 1 7237 Supernat. — — 2 72 37 Cells 0.042 2 2 72 37 Supernat. 0.011 1 3 72 37Cells 0.059 2 3 72 37 Supernat. 0.017 1 4 72 37 Cells 0.077 2 4 72 37Supernat. 0.0043 1

b) Determination According to the Liu Method

The Liu method has been described in Biomed. Biotechnol. 12811, 923(2011).

Table 3 reports data related to the conversion of LA into CLA byLactobacillus paracasei LMG S-26420, according to the method describedby Liu P et al. in Biomed. Biotechnol. 12811, 923, 2011.

The method is based on the determination of the CLA amount in thesupernatant of cultures in MRS® medium containing differentconcentrations of LA from 0.05 to 0.5 mg/ml. The cultures were incubatedfor a period of 24 and 96 hours and at temperature of 30° and 37° C. TheCLA obtained by the conversion of LA was determined with the Barretmethod as described in the Example 1.a.

TABLE 3 LA Cell CLA Conversion concen- Cell incubation concen-percentage tration incubation temperature tration (%) of LA (mg/ml) time(h) (° C.) (mg/ml) into CLA 0.00 24 30 0.001 — 0.05 24 30 0.005 9 0.1 2430 0.005 5 0.2 24 30 0.005 2 0.3 24 30 0.006 2 0.4 24 30 0.006 2 0.5 2430 0.006 1 0.00 96 30 0.001 — 0.05 96 30 0.006 12 0.1 96 30 0.005 5 0.296 30 0.005 3 0.3 96 30 0.006 2 0.4 96 30 0.006 1 0.5 96 30 0.006 1 0.0024 37 0.001 — 0.05 24 37 0.004 8 0.1 24 37 0.005 5 0.2 24 37 0.005 2 0.324 37 0.005 2 0.4 24 37 0.006 1 0.5 24 37 0.006 1 0.00 96 37 0.001 —0.05 96 37 0.005 10 0.1 96 37 0.006 6 0.2 96 37 0.006 3 0.3 96 37 0.0072 0.4 96 37 0.006 2 0.5 96 37 0.008 2

c) Determination According to the Chromatographic Method (HPLC)

The chromatographic method allows the determination of CLA geometricalisomers, the isomer c9-t11, the isomer trans 10-cis12 and the isomertrans9-trans11 in cell suspensions and in supernatants of culturesaccording to the method described in a) and in b) and reported in Tables2 and 3.

The samples were methylated as described by Kramer J. et al. in Am. J.Clin. 79, 1137 S 2004 and the obtained methyl esters were separated bysilver-ion high-pressure chromatography (HPLC). CLA isomers wereseparated using three 4.6 mm×250 mm CHromSpher Lipid columns, connectedin series, with 5μ-sized particles. The isomers were eluted with asolution of hexane-acetonitrile: 99-1 and detected with a diode arraydetector and a UV detector at 234 nm in series. The quantitativedetermination was carried out by using solutions at known concentrationsof CLA isomers.

Table 4a reports the determination of CLA isomers in pellet samplescoming from cultures with LA pre-incubation at a concentration of 0.05mg/ml and incubation of washed cells at different concentrations of LAat 37° C. and an incubation time of 72 hours as reported in a), Ogawamethod.

TABLE 4a CLA CLA CLA Isomers Total isomer isomer isomer (t9-t11 andconversion LA conc. t9-t11 c10-t12 c9-t11 c9-t11)* percentage (mg/ml)(mcg/g) (mcg/g) (mcg/g) (%) (%) 0.05 1.39 2.75 2.07 3.46 14.8 0.01 1.563.50 3.07 4.63 0.2 0.4 1.21 2.78 2.40 3.61 0.2 *isomers with biologicalactivity

Table 4b reports the concentration of CLA isomers in supernatant samplescoming from cultures incubated at different concentrations of LA asreported in b), Liu method.

TABLE 4b CLA isomer Isomers Total CLA isomer CLA isomer CLA isomer(t9-t11 (t9-t11 conversion LA conc. t9-t11 c10-t12 c9-t11 and c9-t11)*and c9-t11)* percentage (mg/ml) (mcg/g) (mcg/g) (mcg/g) (mcg/g) (%) (%)0.05 (24 h) 1.81 5.07 3.30 44.16 46.76 25.9 0.05 (96 h) 2.45 6.33 5.0642.26 46.67 35.5 0.2 (96 h) 1.74 5.64 5.04 46.15 46.15 7.3 0.5 (96 h)1.95 5.80 5.22 7.17 44.67 3.2 *isomers with biological activity

Example 2

Fermentation Process for the Preparation of the Strain Lactobacillusparacasei LMG S-26420.

The fermentations were carried out in a Sartorius fermenter. Theinoculum of the strain Lactobacillus paracasei LMG S-26420 at 1%,corresponding to 40 ml, was cultured in 4 litres of MRS® medium at atemperature of 37° C. for 8 hours.

The cell mass obtained by different fermentations was concentrated bycentrifugation and washed with sterile water. The optic density on theaqueous suspension of the cell mass was determined by spectrophotometricmethod at 625 nm and the Colony Forming Units (CFU) were determined bydecimal plate count.

Some preparations were carried out with different fermentationparameters in order to determine the best bacterial growing conditionsfor the strain Lactobacillus paracasei LMG S-26420.

Table 5 reports the parameters related to fermentations at differentcarbon dioxide pressures, stirring speed and pH. Table 5 also reportsthe results obtained by measuring the optic density and the CFU numberon biomass at fermentation end, after 8 hours.

TABLE 5 Fermentation parameters Prep. 1 Prep. 2 Prep. 3 Prep. 4 Prep. 5Prep. 6 Culture medium MRS ® MRS ® MRS ® MRS ® MRS ® MRS ® + Trealose 1%pH n.c. n.c. n.c. 5.5 5.5 5.5 P-CO2 (bar) 0.8 bar - 0.8 bar - 1 bar - 1bar - n.c. 0.8 bar - 30 min. 30 min. 60 min. 60 min. 45 min. Stirringspeed (rpm) 100 150 150 150 150 150 Biomass weight (g) 40 40 32 35 47 62OD 625 nm - T0 3.50 0.075 0.041 0.045 0.036 0.04 OD 625 nm - 8 ore 0.0871.78 1.406 1.63 1.553 1.50 CFU/ml - T0 3.50E+06 2.50E+07 6.30E+073.30E+07 3.00E+07 1.75E+07 CFU/ml -8 ore 2.30E+09 1.10E+09 1.90E+092.20E+09 2.60E+09 1.05E+09 n.c.: non controlled

The CFU number of Lactobacillus paracasei LMG S-26420 at the end of theculture was comprised in a range from 1.9×10⁹ to 2.6×10⁹ per millilitreof culture and the biomass produced was comprised from 8 to 16 grams perlitre of culture.

The CFU measure was carried out on Petri dish by serial dilution platingof cell mass and microbial colony count.

Cell masses can be maintained at temperatures lower than 0° C. ordirectly lyophilised to be preserved in solid form or used forpharmaceutical/nutritional preparations.

Example 3

Preparation of the Strain Lactobacillus paracasei LMG S-26420 inLyophilic Form.

The product obtained from the preparation described in Example 2(Preparation 6) was lyophilised.

To value the effect of a cryoprotectant on the lyophilisation ofLactobacillus paracasei LMG S-26420, the lyophilisation processes werecarried out in the presence of cyclodextrins, trealose and mannitol.

The cell pellet was suspended in water and an amount of cyclodextrins at10% (p/V) was added.

The solution was divided in three parts:

Solution A: cyclodextrins 10% (w/V)

Solution B: cyclodextrins 10% (w/V)+trealose 20% (w/V)

Solution C: cyclodextrins 10% (w/V)+mannitol 15% (w/V).

Table 6 reports the lyophilisation process parameters for the solutionsA and B.

TABLE 6 STEPS of lyophilisation process (I) Parameters Freezing −50° C.,v = 2° C./min, −50° C. × 120 min Primary drying −50° C. × 15 min, −30°C., v = 0.3° C./min, −30° C. × 300 min P = 100 mTorr Secondary drying30° C., v = 0.16° C./min, 30° C. × 480 min P = 100 mTorr Total Time 14hours

Table 7 reports the parameter of the lyophilisation process for thesolutions C.

TABLE 7 STEPS of lyophilisation process (I) Parameters Freezing −60° C.× 120 min Annealing −10° C., v = 0.55° C./min −10° C. × 240 min −50° C.× 120 min Primary drying −18° C., v = 0.16° C./min −18° C. × 600 min P =120 mTorr Secondary drying 25° C., v = 0.07° C./min P = 50 mTorr

The obtained lyophilic strain Lactobacillus paracasei LMG S-26420 wasstored in bottles or sachets at 4° C.

Example 4

Determination of Glass Transition Temperatures Tg, T′g The glasstransition temperature Tg and T′g of the solutions A and B beforelyophilisation was determined by differential scanning calorimeter,using a Diamond DSC instrument, applying cycles of freezing/heating asreported in Table 8.

TABLE 8 Temperature 1 Temperature 2 Speed Step (° C.) (° C.) (° C./min)1 25 −60 10 2 −60 25 40 3 25 100 10 4 100 −50 50 5 −50 170 40

The cycle reported in Table 9 was used for the solutions C.

TABLE 9 Temperature 1 Temperature 2 Speed Step (° C.) (° C.) (° C./min)1 25 −60 10 2 −60 −10 40 3 −10 −10 Constant for 120 min 4 −10 −60 40 5−60 25 40

Glass transition temperatures values of solutions Tg and of frozenaqueous solutions, T′g of the solutions A, B and C to be lyophilised arereported in Table 10.

TABLE 10 Lyophilisation T′g Tg LMG S-26420 Preparation (° C.) (° C.)Solutions A = cyclodextrins A-1 −12.25 148.55 10% (p/V) A-2 −12.75140.81 A-3 −13.05 144.44 B = cyclodextrins 10% B-1 −26.50 112.00 (p/V) +Trealose 20% (pN) B-2 −26.63 114.64 B-3 −26.54 117.54 B-4 −26.75 112.45B-5 −26.54 114.40 B-6 −30.58 111.93 B-7 −34.76 116.629 B = cyclodextrins10% B-8 −27.96 111.77 (p/V) + Trealose 20% (pN) B-9 −27.50 118.40 C =cyclodextrins 10% C-1 T′g¹: −36.8 n.d. (p/V) + mannitol 15% T′g²: −28  T′g³: −14.8 C-2 −28.4 106.00

Example 5

Characterization of the Lyophilic Strain Lactobacillus paracasei LMGS-26420.

a) Strain Viability Determination

To determine the yield and viability of the strain Lactobacillusparacasei LMG S-26420 after the lyophilisation process of the solutionsA, B and C prepared according to Example 3, the CFUs of the strain weredetermined before and after the lyophilisation process, by Petri dishcount. The determination was made on an amount of lyophilic productcorresponding to 0.5 grams, suspended in 49.5 grams of dilution buffer(MRS®) and diluted as serial dilution until reaching those useful forthe count. The diluted product was then deposited on a Petri dishcontaining 20 mL of MRS® agar medium, previously sterilized inautoclave, and incubated at 37° C. in anaerobiosis for 72 hours.

The determination was carried out on many lyophilic preparations madefrom the solutions A, B and C and the results are reported in Table 11.

TABLE 11 Yield Lyo- percentage philisation Prep- (%) lyo- LMG ara-Before-lyo After-lyo (T₀) philisation S-26420 tion UFC/ml ± SD UFC/g ±SD process Solution A = A-1 4.6 ± 0.6 × 10¹⁰ 5.6 ± 0.4 × 10¹⁰ 13.9 ±0.9  cyclodextrins A-2 4.0 ± 0.5 × 10¹⁰ 3.4 ± 0.2 × 10¹⁰ 16.6 ± 1.1  10%(wN) A-3 2.8 ± 0.5 × 10¹⁰ 3.3 ± 0.5 × 10¹⁰ 15.8 ± 2.9  B = B-1 3.2 ± 0.5× 10¹⁰ 2.9 ± 0.7 × 10¹⁰ 26.2 ± 6.5  cyclodextrins B-2 4.4 ± 0.1 × 10¹⁰3.3 ± 0.3 × 10¹⁰ 22.8 ± 1.9  10% (wN) + B-3 3.3 ± 0.1 × 10¹⁰ 6.3 ± 0.3 ×10¹⁰   57 ± 27.0 Trealose B-4 2.2 ± 0.8 × 10¹⁰ 3.9 ± 0.3 × 10¹⁰ 45.1 ±2.7  20% (wN) B-5 2.8 ± 0.2 × 10¹⁰ 4.6 ± 0.9 × 10¹⁰ 41.7 ± 7.5  B-6 3.6± 0.1 × 10¹⁰ 3.7 ± 0.5 × 10¹⁰ 23.3 ± 2.9  B-7 3.1 ± 0.9 × 10¹⁰ 2.9 ± 0.8× 10¹⁰ 21.9 ± 6.0  B-8 5.5 ± 0.7 × 10¹⁰ 1.4 ± 0.8 × 10¹⁰ 35.8 ± 1.4  B-94.3 ± 0.9 × 10¹⁰ 3.7 ± 0.8 × 10¹⁰ 22.6 ± 2.1  C = C-1  7.3 ± 2. × 10¹⁰<1 × 10¹⁰ 13.9 ± 0.9  cyclodextrins C-2 3.6 ± 0.5 × 10¹⁰ 2.3 ± 0.8 ×10¹⁰ 9.56 ± 4.0  10% (p/V) + mannitol 15%

Table 11 confirms the effect of trealose during the lyophilisationprocess, leading to a recovery higher than 30% in CFU.

Mannitol can be added to trealose if a bigger lyophilic mass is desired,in particular for small dosages of Lactobacillus paracasei LMG S-26420.

Example 6

Determination of Water Activity and Weight Loss by Drying LyophilicLactobacillus paracasei LMG S-26420.

a) Determination of the Water Activity (a_(w))

The determination of the water activity corresponds to the determinationof free water in lyophilic preparations of Lactobacillus paracasei LMGS-26420 of the solutions A, B and C prepared following Example 3. Thedetermination was carried out by placing a sample of about 1 gram ofeach lyophilic product in an Aqualab VSA Decagon instrument thatmeasures water activity of a sample with a humidity dielectric detector,directly giving the measure that is directly proportional to the watermoles over the sample moles and the obtained values are reported inTable 12.

TABLE 12 Lyophilisation components Preparation a_(w) LMG S-26420 No.(water activity) Solution A = cyclodextrins A-1 n.d 10% (wN) A-2 n.d.A-3 0.358 B = cyclodextrins 10% (wN) + B-1 n.d Trealose 20% (wN) B-2 n.dB-3 0.363 B-4 0.153 B-5 0.100 B = cyclodextrins 10% (w/V) + B-6 0.120Trealose 20% (w/V) B-7 0.136 B-8 0.166 B-9 0.087 Solution C =cyclodextrins 10% C-1 0.174 (w/V) + mannitol 15% (w/V) C-2 0.134

All obtained lyophilic have a_(w) values lower than 0.6 confirming thatthey can be stored for long periods without risks of degradation.

b) Determination of the Loss of Water Content (LOD)

The humidity content of the lyophilic samples was obtained using athermoscales Mettler and values are reported in Table 13.

TABLE 13 Humidity Lyophilisation components Preparation Percentage LMGS-26420 No. LOD (%) Solution A = cyclodextrins A-1 3.61 10% (w/V) A-22.58 A-3 3.9 B = cyclodextrins 10% (wN) + B-1 4.01 Trealose 20% (wN) B-24.24 B = cyclodextrins 10% (wN) + B-3 4.90 Trealose 20% (wN) B-4 3.39B-5 2.94 B-6 2.85 B-7 3.05 B-8 3.21 B-9 2.41 Solution C = cyclodextrins10% C-1 2.14 (w/V) + mannitol 15% (wN)

Example 7

Determination of Stability of Lyophilic Preparations of the StrainLactobacillus paracasei LMG S-26420.

The lyophilic preparations of the strain Lactobacillus paracasei LMGS-26420 were placed at 4° C. and their stability was determined bymeasuring the bacterial charge and the water activity (a_(w)) (TO), attime zero, 3 and 6 months. The obtained results are reported in Table14.

TABLE 14 Yield a_(w) (water a_(w) (water CFU/g ± SD CFU % activity)activity) Preparation T0 T3 T6 T6/T0 T0 T6 A-1 3.4 ± 0.2 × 10¹⁰ 1.3 ±0.2 × 10¹⁰ 9.8 ± 5.7 × 10⁴ <10% A-2 3.5 ± 0.6 × 10¹⁰ 6.4 ± 1.5 × 10⁹ <1.78 × 10² <10% A-3 2.9 ± 0.7 × 10¹⁰ 7.1 ± 2.5 × 10⁹  — B-1 2.9 ± 0.7 ×10¹⁰ 7.1 ± 2.5 × 10⁹  — B-2 3.3 ± 0.3 × 10¹⁰ 1.2 ± 0.1 × 10¹⁰ — 0.1530.276 B-3 6.3 ± 0.2 × 10¹⁰ 1.9 ± 0.2 × 10¹⁰ 1.8 ± 0.1 × 10¹⁰ 27.7 ± 1.00.100 0.163 B-4 3.9 ± 0.2 × 10¹⁰ 2.3 ± 0.6 × 10¹⁰ 2.9 ± 0.7 × 10¹⁰  75.0± 19.2 0.120 0.313 B-5 4.6 ± 0.9 × 10¹⁰ 4.9 ± 0.4 × 10¹⁰ — 0.136 0.279B-6 3.7 ± 0.5 × 10¹⁰ 4.2 ± 0.3 × 10¹⁰ 5.0 ± 0.6 × 10¹⁰ 136.2 ± 14.80.143 0.182 B-7 2.9 ± 0.8 × 10¹⁰ 1.9 ± 0.3 × 10¹⁰ 2.6 ± 2.1 × 10¹⁰  91.1± 17.9 0.087 —

The table shows that the lyophilic products obtained in the presence oftrealose are stable at 4° C. for 6 months and that the water activity(a_(w)) is always lower than 0.6.

Example 8

Compositions Comprising Lactobacillus paracasei LMG S-26420 in Sachets

The preparations in sachets comprise an amount of lyophilic product,prepared as in Example 2 (preparations B), of 800 mg and 500 mg,corresponding respectively to about 3×10¹⁰ and 2×10¹⁰ living cells ofLactobacillus paracasei LMG S-26420.

The lyophilic product was mixed with excipients, preventively sieved,and the homogeneous mixture was divided in sachets. The unitarycompositions are reported in Table 15.

Among the described compositions, one comprises inuline, anothercomprises xilo-oligosaccharide and the others a mixture offructo-oligosaccharides formed by a chain of fructose molecules bound toa molecule of glucose (Actilight 950P).

TABLE 15 Comp. 1 Comp. 2 Comp. 3 Comp. 4 weight weight weight weightComponent (mg) (mg) (mg) (mg) Lactobacillus paracasei 800 800 500 500lyophilic LMG S-26420 Inulin 3200 Xilo-oligosaccharide 3200 506 506Flavour (Passion fruit) 44 44 Silica 10 10 Astaxantine 160 Actilight950P 2940 1720 Total weight 4000 4000 4000 4000

The compositions can comprise vitamins, like for example vitamin E,vitamin B1, vitamin B2, vitamin B16.

The compositions in sachets can be suspended in water solutions or insemi-solid foods.

Example 9

Compositions Comprising Lactobacillus paracasei LMG S-26420 in Tablets

The preparations in tablets containing an amount of 250 mg of lyophilicproduct, corresponding to about 1×10¹⁰ living cells of Lactobacillusparacasei LMG S-26420, were obtained by mixing a lyophilic withexcipients preventively sieved on a net of 800 micron. The mixture wasthen compressed in a Ronchi compressing machine or an analogous machineby applying a compressing force of about 13 KN.

The unitary composition of the tablets is reported in Table 16.

TABLE 16 Components Amount (mg) Lactobacillus paracasei 250 (1 × 10¹⁰ ±CFU) LMG S-26420 Isomalto 278.0 Croscarmellose 9.9 Talc 5.5 Silica 3.8Magnesium stearate 2.8 Total weight 550

1. A bacterial strain belonging to the species Lactobacillus paracaseifiled at the Coordinated Collection of Microorganism-BCCM/LMG BacteriaCollection, Microbiology Laboratory-Ghent University, registered withthe number LMG S-26420, characterized in that it converts linoleic acidin conjugated linoleic acid in a percentage higher than 30%.
 2. Abacterial strain Lactobacillus paracasei LMG S-26420 according to claim1, characterized in that it converts linoleic acid into the isomers withbiological activity of conjugated linoleic acid c9-t11 e t9-t11, whereintheir sum is in a percentage higher than 30% if compared to the otherisomers.
 3. A process for the production of the strain Lactobacillusparacasei LMG S-26420 of claim 1 characterized by the following steps:inoculating the strain Lactobacillus paracasei LMG S-26420 in avolumetric percentage from 0.1% to 10%, in a suitable culture medium ata temperature from 30° C. to 37° C. at pH values between 4.5 and 7.5 fora time from 6 to 15 hours; separating the bacterial mass from theculture broth by centrifugation; storing the bacterial mass at atemperature lower than 0° C., or lyophilising.
 4. A process for theproduction of the bacterial strain Lactobacillus paracasei LMG S-26420according to claim 3, comprising the step of lyophilizing in thepresence of cryoprotectants selected among soluble carbohydrates.
 5. Aprocess according to claim 4 for the production of the lyophilicbacterial strain Lactobacillus paracasei LMG S-26420 in the presence oftrealose.
 6. A process according to claim 3, wherein the strainLactobacillus paracasei LMG S-26420 is obtained with a number of colonyforming units higher than 109 for millilitre of culture, and the biomassin an amount higher than 4 grams per litre of culture.
 7. A nutritionalor food or pharmaceutical composition comprising a cell amount of thebacterial strain Lactobacillus paracasei LMG S-26420 from 1×108 to5×1011, together with acceptable excipients in form of tablets, granulesfor sachets, capsules or liquid suspensions.
 8. A nutritional or food orpharmaceutical composition according to claim 7 comprising prebiotics,vitamins, mineral salts.
 9. A nutritional or food or pharmaceuticalcomposition according to claim 8, wherein the prebiotics are selectedfrom the group consisting of fructo-oligosaccharides, inulines,galacto-oligosaccharides, xylo-oligosaccharides, andisomalto-oligosaccharides and the vitamins are selected from the groupconsisting of vitamins E and B.
 10. A nutritional or food orpharmaceutical composition according to claim 7 comprisingbifidobacteria.
 11. Method of treating or preventing pathologies ordiseases related to a deficiency of conjugated linoleic acid and in allcases where a probiotic is beneficial in a subject in need thereof, saidmethod comprising administering to said subject a pharmaceuticaleffective amount of a nutritional or food or pharmaceutical compositioncomprising the bacterial strain Lactobacillus paracasei LMG S-26420. 12.Method of treating and/or preventing inflammatory diseases, intestinalinflammatory pathologies, oxidative stress, tumour, autoimmune diseases,diabetes and atherosclerosis in a subject in need thereof with anutritional or food or pharmaceutical composition comprising thebacterial strain Lactobacillus paracasei LMG S-26420, said methodcomprising: administering to said subject a pharmaceutical effectiveamount of said nutritional or food or pharmaceutical compositioncomprising the bacterial strain Lactobacillus paracasei LMG S-26420. 13.Method of treating diseases related to bacterial dysbiosis in a subjectin need thereof with the nutritional or food or pharmaceuticalcomposition according to claim 7 comprising lactobacilli belonging todifferent strains and/or Bifidobacteria, said method comprising:administering to said subject a pharmaceutical effective amount of saidnutritional or food or pharmaceutical.
 14. Method of increasing bodymass and thermogenesis in a subject in need thereof with a nutritionalor food or pharmaceutical composition comprising the bacterial strainLactobacillus paracasei LMG S-26420, said method comprising:administering to said subject a pharmaceutical effective amount of saidnutritional or food or pharmaceutical composition comprising thebacterial strain Lactobacillus paracasei LMG S-26420.